title: Impact of 7-ketocholesterol and very long chain fatty acids on oligodendrocyte lipid membrane organization: evaluation via LAURDAN and FAMIS spectral image analysis.
abstract: In the context of multiple sclerosis and X-linked adrenoleukodystrophy, 7-ketocholesterol (7KC) and very long chain fatty acids (C24:0, C26:0) are supposed to induce side effects respectively on oligodendrocytes which are myelin (which is a lipoproteic complex) synthesizing cells. The effects of 7KC (25, 50 μM), C24:0 and C26:0 (10, 20 μM) on cell viability and lipid membrane organization were investigated on 158N murine oligodendrocytes. Concerning 7KC and fatty acids (at 20 μM only): 1) cell growth was strongly inhibited; 2) marked induction of cell death was revealed with propidium iodide (PI); 3) no apoptotic cells were found with C24:0 and C26:0 (absence of cells with condensed and/or fragmented nuclei, of FLICA positive cells and of PI negative/SYTO16 negative cells); 4) some apoptotic cells were detected with 7KC. Fatty acids (at 20 μM only) and 7KC also induced a disorganization of lipid membranes revealed with Merocyanine 540. So, to point out the effects of 7KC (25 μM), C24:0 and C26:0 (20 μM) on the lateral organization of lipid membranes, we used LAURDAN, which gives simultaneous information about morphology and phase state of lipid domains: its emission is blue in the ordered lipid phase, green in the disordered lipid phase. To overcome the qualitative filtering settings of blue and green emission colors, data obtained by mono- and bi-photon confocal microscopy were analyzed by spectral analysis. Sequences of emission images were obtained on both mono- and bi-photon confocal microscopes and processed by means of Factor Analysis of Medical Image Sequences (FAMIS), which is a relevant tool to unmix emission spectra and provide pure color images. Only 7KC was capable to induce a green emission with LAURDAN. Thus, at concentrations inducing oligodendrocyte cell death, 7KC (25 μM) is more efficient than C24:0 and C26:0 (20 μM), to trigger lateral lipid membrane disorganization.
authors.affiliation: INSERM U678/UMR-S UPMC, IFR 14, CHU Pitié-Salpêtrière, 75634 Paris Cedex 13, France. kahn@imed.jussieu.fr
authors.forename: Edmond
authors.initials: E
authors.lastname: Kahn
authors.forename: Mauhamad
authors.initials: M
authors.lastname: Baarine
authors.forename: Aurélien
authors.initials: A
authors.lastname: Dauphin
authors.forename: Kévin
authors.initials: K
authors.lastname: Ragot
authors.forename: Nicolas
authors.initials: N
authors.lastname: Tissot
authors.forename: Alix
authors.initials: A
authors.lastname: Seguin
authors.forename: Franck
authors.initials: F
authors.lastname: Ménétrier
authors.forename: Zilal
authors.initials: Z
authors.lastname: Kattan
authors.forename: Claude-Marie
authors.initials: CM
authors.lastname: Bachelet
authors.forename: Frédérique
authors.initials: F
authors.lastname: Frouin
authors.forename: Gérard
authors.initials: G
authors.lastname: Lizard
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005227:Fatty Acids / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D007653:Ketocholesterols / Q000494:pharmacology*; D007848:Laurates / Q000494:pharmacology*; D008297:Male; D008563:Membrane Lipids / Q000737:chemistry*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D009836:Oligodendroglia / Q000187:drug effects / Q000648:ultrastructure; D047391:alpha-Cyclodextrins / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D005227:Fatty Acids; D005456:Fluorescent Dyes; D007653:Ketocholesterols; D007848:Laurates; D008563:Membrane Lipids; D047391:alpha-Cyclodextrins; D015081:2-Naphthylamine; C003001:7-ketocholesterol; C065580:laurdan; C032613:alpha-cyclodextrin